Zinc is an essential nutrient but also potentially toxic if accumulated to excess. Therefore, cells have evolved with efficient mechanisms to maintain zinc homeostasis. In addition, cells can alter specific metabolic processes to adapt to the stress of zinc deficiency. Among eukaryotes, these mechanisms of zinc homeostasis and low zinc adaptation are best understood in the yeast Saccharomyces cerevisiae. In yeast, the Zap1 transcription factor is the central regulator of zinc homeostasis. Zap1 activates expression of its target genes in zinc-limited cells and its activity is repressed in replete cells. Over the years, synergistic approach of characterizing the molecular mechanisms of Zap1's zinc responsiveness, combined with analyzing the function of Zap1 target genes, has led to many key discoveries about how cells survive the stress of zinc deficiency. In this application, two specific aims are proposed. In Specific Aim 1, the Zap1 transcription factor will be further characterized. New Zap1 target genes will be identified and candidate genes previously identified by transcriptomics will be verified as bona fide targets. The role of Zap1 autoregulatio in establishing the regulatory hierarchy of Zap1 target genes will be determined and the mechanism regulating Zap1 DNA binding in response to zinc will be dissected. In Specific Aim 2, the function of key Zap1 target genes in zinc-limited cells will be characterized. A focus of th next funding period is the Tsa1 chaperone. Functional genomics studies and specific analysis of Tsa1 have indicated that zinc-deficient cells experience a crisis of protein homeostasis. The source of unfolded proteins in zinc-limited cells will be determined as will the mechanisms of protein folding, degradation, and sequestration that cells use to survive. Preliminary results also indicate that Tsa1 orthologs in humans are critical for low zinc growth and their roles in human cells will also be investigated. Health relevance: The long-term goal of the proposed research is to determine how cells respond and adapt to the stress of zinc deficiency, a common nutrient deficiency in the US and the world. The research also explores the effect of zinc deficiency on protein folding and this work may ultimately lead to fundamental insights into diseases of protein misfolding such as amyotrophic lateral sclerosis (ALS), Parkinson's, Alzheimer's, and prion diseases and their relationships with metal homeostasis.